The biosynthesis of glycosylphosphatidylinositol (GPI)-anchored proteins (such as the folate receptor, prion protein, and the neural cell adhesion molecule (NCAM)) is critical for normal cell growth, and perturbed in human cancers as well as a number of genetic diseases. The latter include an inherited GPI deficiency characterized by venous thrombosis and seizures, and paroxysmal nocturnal hemoglobinuria (PNH), an acquired hemolytic disease that is caused by a defect in the first step of GPI biosynthesis in multipotent hematopoietic human stem cells. Genetic abrogation of GPI biosynthesis results in embryonic lethality in mammals. The GPI assembly pathway has been validated as a therapeutic target for protozoal and fungal diseases, including African sleeping sickness and the life-threatening opportunistic fungal infections that afflict immuno-compromised individuals. The aims of this proposal are to understand aspects of the biosynthesis of GPI-proteins with the overall objective of developing strategies to manipulate and control the GPI pathway. Such efforts are central to the development of anti-protozoal and anti-fungal drugs, as well as to the treatment of cancer and inherited diseases in which GPI-anchored proteins play a key part. We are interested in two critically important steps of GPI-protein biosynthesis: the attachment of a GPI anchor to protein and the enigmatic flip-flop of GPI lipids across the endoplasmic reticulum (ER) membrane. GPI transamidase (GPIT), the enzyme that attaches GPI anchors to protein, is a poorly understood membrane-bound hetero-pentameric complex. Genes encoding three of the GPIT subunits have been recently identified as oncogenes, raising the possibility that the GPI pathway may provide a novel target for human cancer treatment. Our aim is to define the functional role of GPIT's many subunits;we are especially interested in the oncoproteins PIG-U and GAA1 that we hypothesize to be the GPI binding elements of the complex. We also propose analyses of trypanosome GPIT that will, in the long term, yield results pertinent to the design of reagents that could be used to selectively block essential GPI anchoring in trypanosomatid protozoa while these parasites are resident in their mammalian hosts. Our second aim is to identify GPI flippase, the novel transporter that is required to translocate (flip) GPI lipid intermediates across the ER membrane during GPI assembly. GPI flipping is an obligatory step in GPI assembly and the only one that currently remains to be biochemically and genetically defined. Since no ER lipid flippase of any type has been functionally identified, our aim to identify the GPI flippase will not only contribute specifically to an understanding of GPI assembly but will also provide fundamental information on lipid translocation events in the ER in general.